The multivariate models were fitted using a generalized linear model (R-package GLM) prior ROC analysis (R-package pROC). EDTA plasma was provided by Atlas Antibodies AB, Sweden and originated from three males and three females all without known disease diagnosis. This mixture of plasma was diluted 1:300 in assay buffer and heat treated as above. Antibodies HPA-1, MAB-1.1 and normal rabbit IgG (CAB-5) and normal mouse IgG (sc-2025, Santa Cruz Biotechnology) were coupled

to Luminex beads as above, and beads carrying the different antibodies were incubated separately with 1 ml of heat treated plasma samples overnight at RT on gentle rotation. After incubation beads were washed 3× in PBS/0.03% CHAPS (Sigma) find more and 2× in 0.03% Selleck NVP-BGJ398 CHAPS, to be then re-suspended in 50 μl ammonium bicarbonate 50 mM to perform on beads trypsin digestion. Captured proteins were reduced with dithiothreitol (DTT) 1 mM and alkylated by iodoacetamide (IAA) 4 mM. Alkylation was quenched adding 1 mM DTT. Proteins were digested by trypsin (Promega) overnight at 37 °C and peptides were separated from beads, dried and re-suspended in buffer A (97% water, 3% acetonitrile (ACN), 0.1% formic acid (FA)). In each LC–MS run, the LC auto sampler (HPLC 1200 system, Agilent Technologies) injected 5 μl of sample into a C18 guard desalting column (Zorbax 300SB-C18,

5 mm × 0.3 mm, 5 μm bead size, Agilent). We then used a 15 cm long C18 picofrit column (100 μm internal diameter, 5 μm bead size, Nikkyo Technos Co., Tokyo, Japan) installed on to the nano electrospray ionization (NSI) source. Solvent A was 97% water, 3% acetonitrile (ACN), 0.1% formic acid (FA); and solvent B was 5% water, 95% ACN, 0.1% FA. At a constant flow of 0.4 μl/min, the linear gradient went from 2% B up to 40% B in 45 min, followed by a steep increase to 100% B in 5 min. Online LC–MS was performed using a hybrid

LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific). Precursors were isolated with a 2 m/z window. We enabled “preview mode” for FTMS master scans, which proceeded at resolution of 30,000 (profile mode). Data-dependent MS/MS (centroid mode) CYTH4 followed in two stages: firstly, the top 5 ions from the master scan were selected for collision induced dissociation (CID, at 35% energy) with detection in the ion trap (ITMS); and after, the same 5 ions underwent higher energy collision dissociation (HCD, at 37.5% energy) with detection in the orbitrap (FTMS). All MS/MS spectra were searched by Sequest under the software platform Proteome Discoverer (PD, v1.3.0.339, Thermo Scientific) using a target-decoy strategy. The reference database used was the human reference proteome set from uniprot.org (2013-04-18). Precursor mass tolerance of 10 ppm and product mass tolerances of 0.02 Da for HCD-FTMS and 0.36 Da for CID-ITMS were used.